This invention is directed to a method of synthesis of oligonucleotides having low molecular weight tail molecules joined to the 3'-terminus of the oligonucleotide via a linking molecule, to oligonucleotides having low molecular weight tail molecules joined to their 3'-terminus and to intermediates utilized for the synthesis of such oligonucleotides.
Oligonucleotides have various uses including acting as primers for polymerase chain reaction synthesis of DNA. Oligodeoxynucleotides (abbreviated as ODN) are conveniently synthesized on solid phase supports using phosphite-triester synthetic methods. A detailed review of such syntheses was published in Atkinson, T., Smith, M. (1984) in Oligonucleotide Synthesis, A Practical Approach, Gait, M. J. (ed.), IRL Press, pp. 35-81. This review gives detailed step by step conditions for the practical synthesis of oligonucleotides. Indeed, methods as outlined in this review are presently utilized in commercial oligonucleotide synthesizers available from various manufacturers.
Asseline et al., Proc. Natl. Acad. Sci. 81:3297-3301 (1984), describes the synthesis of certain oligonucleotides wherein an intercalating agent was covalently linked to the oligodeoxynucleotides. The intercalating agent utilized was 2-methoxy-6-chloro-9-aminoacridine. The acridine molecule was covalently linked to an oligodeoxynucleotide via a methylene chain of from 3 to 6 carbon atoms connecting the 3'-phosphate of the oligodeoxynucleotide to the 9-amino group of the acridine. The authors of this study found that the acridine modified oligonucleotides in the presence of a complementary sequence showed strong stabilization by the intercalating agent, i.e., the acridine. These authors measured certain thermodynamic parameters and showed via these parameters that the covalent attachment of the acridine ring strongly stabilized the binding of a synthetic oligonucleotide to its complementary sequence. The melting temperature, i.e., T.sub.m, of an oligonucleotide having the intercalating agent attached thereto and its complementary strand was increased compared to the melting temperature of a similar oligonucleotide not bearing the intercalating agent thereon and its complementary strand. The authors concluded that the results clearly show that the presence of the intercalating agent strongly stabilized the complex formed between an oligonucleotide and its complementary strand.
In a similar study Letsinger et al., Proc. Natl. Acad. Sci. 86:6553-6556 (1989), prepared a family of oligonucleotides that had a cholesteryl group covalently joined at the 3'-terminal internucleoside phosphate linkage. Oligomers of various length were synthesized. Those bearing the cholesteryl moiety adjacent either the 3' terminus or both the 3' and the 5' terminus were compared to oligomers that were not so substituted. These compounds were tested as to their inhibitory action on HIV-1 replication. Anchoring of a single cholesteryl fragment adjacent to the 3' terminus of a 20-mer oligonucleotide significantly enhanced the antiviral activity of the oligonucleotide. Anchoring of the second cholesteryl fragment at the 5' terminus of the oligonucleotide detracted and led to a reduction of activity compared to the monocholesteryl derivatized oligomer.
The compounds of Letsinger et al. were prepared by first manually preparing a support bound dinucleoside hydrogen phosphonate derivative. A cholesteryl group was then tethered to the internucleoside phosphorus by oxidative phosphoramidation. The oligonucleotide was elongated from the original dinucleotide on a commercial DNA synthesizer using phosphoramidite chemistry.
Controlled pore glass beads for use in commercial oligonucleotide synthesis are available from CPG, Inc., Pierce Chemical Co. and Sigma Chemical Co. As is described in Atkinson and Smith, above, the controlled pore glass beads, hereinafter alternately referred to as CPG's, are derivatized by the manufacturers with a long chain alkylamine group, such that a free amino group is available at the end of the long chain alkylamine that in turn is attached to the CPG. Various amide linkages can be formed with the terminal amine of the long chain alkylamine on the CPG's for attachment of a growing oligonucleotide during synthesis of the same. An improvement of this synthesis was reported by Pon et al., BioTechniques 6(8):768 (1988). In this report Pon et al. precap a suspected contaminate side group on the long chain alkylamine CPG and introduce the use of DEC (a water soluble carbodiimide, i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or EDC) in place of the more commonly used (and toxic) linking agent DCC, i.e., dicyclohexylcarbodiimide.
Nelson et al., Nuc. Acids Research 17(18):7187-7194 (1989), recently described the synthesis of an oligonucleotide incorporating a 3'-terminal substituent thereon. To prepare a 3'-tailed oligonucleotide, Nelson et al. derivatized the secondary hydroxyl of N-Fmoc-O-DMT-3-amino-1,2-propanediol by treating with succinic anhydride in the presence of DMAP (dimethylaminopyridine), and then subsequently treated with p-nitrophenol in DCC. The activated derivative was then anchored to a long chain alkylamine CPG support. The dimethoxytrityl blocking group was removed from the primary alcohol of the propanediol and an oligonucleotide was synthesized stepwise from the primary hydroxyl group while supported on the CPG support. The synthetic oligonucleotide was deprotected and cleaved from the CPG support. However, the purity of this 3'-amine-modified oligonucleotide was not demonstrated. At this juncture in this synthesis, the 3'-terminal tail substituent has yet to be coupled to the oligonucleotide. The crude oligonucleotide was biotinylated with a "Biotin-XX-NHS" ester. After biotinylation, a second purification was necessary by both Sephadex and by HPLC. No yield data were given.
The above procedure of Nelson et al. gives an oligonucleotide with an amine functional group that may be derivatized (modified) with a "tailing reagent", then repurified. However, the derivation with the "tailing reagent" is effected only after the synthesis of the oligonucleotide is complete. By effecting the derivation on a completed oligonucleotide, precious oligonucleotide that has been systematically stepwise assembled, nucleotide by nucleotide, can be lost to incomplete reaction, side reactions and/or multiple purifications necessary after the derivation. Additionally, this synthesis did not take advantage of the above Pon et al. improvement.